1. Field of the Invention
This invention pertains to chemiluminescent compounds, their use in assays for the detection of proteases, and kits comprising the compounds and other elements used in protease detection assays. Specifically, dioxetane compounds bearing a proteolytic enzyme-specific amino acid or peptide which, when removed by enzymatic reaction, causes the dioxetane to decompose and chemiluminesce, is provided. These compounds, when added to a sample suspected of containing the protease, provide a rapid, ultrasensitive and convenient means for detecting the presence of the protease in the sample. The amount of light generated, or degree of chemiluminescence, can be correlated with the amount of protease present.
2. Background of the Invention
Applications naming one or more of the inventors herein, as inventors, and assigned to Tropix, have clearly established 1,2-dioxetanes as chemiluminescence compounds which can be used as reporters in ultrasensitive assays that can be conducted quickly, without resort to exotic conditions or elaborate apparatus, for the detection of a variety of biological materials. Among these are U.S. Pat. Nos. 4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; 5,145,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900 and 5,336,596. Other patents commonly assigned with this application have issued, and still other applications are pending. Together, this wealth of patent literature addresses 1,2-dioxetanes, stabilized by a typically polycyclic group, preferably spiroadamantane bonded to one of the carbons of the dioxetane ring, and a moiety bonded to the remaining carbon of the dioxetane ring which is electron sensitive, such that deprotection of the electron sensitive moiety, typically an aryl group, more preferably a phenyl or naphthyl group, leads to an anion, generally an oxyanion, which is unstable, and decomposes. Through decomposition, the O--O bond is broken, and a photon is generated. The same carbon atom to which this electron sensitive moiety is bonded may bear an alkoxy or other electron-active group. Methoxy is a preferred moiety.
The first of the dioxetanes of this class commercialized was 3-(4-methoxy-spiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1.sup.3,7 ]decan]-4-yl)phenyl phosphate, particularly the disodium salt, generally known as AMPPD. This compound has been commercialized by Assignee of this application, Tropix, Inc., as well as a company of Detroit, Mich., Lumigen, Inc. The primary inventor of Lumigen, A. Paul Schaap, has been granted several patents on related technology.
Superior performance can be obtained by selective substitution on the spiroadamantane ring. Substitution, at either bridgehead carbon, with an electron-active species, such as chlorine, improves reaction speed and signal-to-noise ratio (S/N). The chlorine-substituted counterpart of AMPPD, CSPD, has been widely commercialized by Tropix, Inc. of Bedford, Mass. "Third-generation" dioxetane compounds of similar structure, wherein the phenyl or naphthyl moiety also bears an electron-active substituent, such as chlorine, offer further improvements in performance, and have been commercialized by Tropix, Inc. The phosphate moieties is available under the trademarks CDP and CDP-Star.
A common characteristic of all these dioxetanes is the blocking or masking group on the phenyl or naphthyl moiety. Groups which are enzyme-specific substrates are employed, such that, when admixed with the enzyme, the blocking group is removed by the enzyme, leaving an electron rich oxygen moiety attached to the phenyl or naphthyl substituent. Typically, this blocking group has been a phosphate, although other blocking groups, such as a galactoside have also been used. Representative blocking groups are set forth in the patents listed above, which are incorporated herein by reference. These blocking groups have been substrates for enzymes which are specific for the blocking group, the enzymes either being selected as enzymes of interest or potential interest in a biological fluid, or non-endogenous enzymes which may be coupled to a particular target moiety of the sample, their triggering of the dioxetane to generate light being thus evidence of the analyte in the sample to which the non-endogenous enzyme is coupled. Alkaline phosphatase has been the dominant enzyme of interest as a triggering agent.
The existing literature on dioxetanes does not describe a triggerable dioxetane, that is, a dioxetane which can be "deprotected" to induce decomposition and chemiluminescence that can be used to detect, or be triggered by, proteolytic enzymes, that is, peptidases. These enzymes are involved in the life cycle of proteins. Further, proteolytic enzymes are also involved in the processing of proteins, hormones, receptors, growth factors, fertilization, activation of regulatory proteases involved in blood coagulation, fibrinolysis and the complement reaction, and other cellular functions. Thus, the presence or absence of a particular proteolytic enzyme in a biological sample may well indicate the presence or absence of a particular disease state, pathogenic condition or organic syndrome. Families of proteolytic enzymes include serine proteases (chymotrypsin and subtilisin), cysteine proteases (papain), aspattic proteases (penicillopepsin) and metalloproteases (carboxypeptidase and thermolysin).
In addition to being of particular interest as organic moieties that constitute diagnostic markers, protease enzymes are also of considerable interest as enzyme labels, and are used in detergent production and leather processing. Examples of diagnostic protease markers include cathepsin B (cancer), cathepsin G (emphysema, rheumatoid arthritis, inflammation), cathepsin L (cancer), elastase (emphysema, rheumatoid arthritis, inflammation), plasminogen activator (thrombosis, chronic inflammation, cancer), and urokinase (cancer). The use of alkaline proteases in detergents is expected to increase. Assays for protease detection are therefore needed to monitor protein stability in various biological and commercial processes.
Known protease assay conditions are listed in the following table:
______________________________________ Protease Assay Conditions ______________________________________ acylaminoacyl peptidase Ac--Ala--p-nitroanilide in Tris HCl, pH 7.5, 37.degree. C. aminopeptidase M L--Leu--p-nitroanilide in 60 mM Na phosphate buffer, pH 7-7.5, 37.degree. C. cathepsin B Bz--Phe--Arg--NMec, pH 5.5-6.0, 37.degree. C. cathepsin G MeO--Suc--Ala--Ala--Pro--Phe--p- nitroanilide, pH 7.5, 25.degree. C. cathepsin L Cbz--Phe--Arg--NMec in 340 mM Na acetate, 60 mM acetic acid, 4 mM disodium EDTA, pH 5.5, 8 mM dithiothreitol, 30.degree. C. elastase Suc--(Ala).sub.3 -p-nitroanilide, 0.05% Triton X-100 in plastic tubes, pH 7.8, 25.degree. C. subtilisin denatured hemoglobin degradation, pH 7-8 plasminogen activator Val--Leu--Lys--p-nitroanilide in 50 mM Tris-HCl, pH 8.5, 37.degree. C. urokinase Val--Phe--Lys--p-nitroanilide in 50 mM Tris-HCl, pH 8.5, 37.degree. C. ______________________________________
Some of the known and potential applications for proteolytic enzymes are listed in the following table:
______________________________________ Protease Applications ______________________________________ Aminopeptidase detection of gram-neg bacteria Cathepsin B cancer marker Cathepsin G emphysema, rheumatoid arthritis, inflammation marker Cathepsin L cancer marker Elastase emphysema, rheumatoid arthritis, inflammation marker Subtilisin used in leather processing as a depilatory, detergents, protein hydrolysate production (e.g. Optimase and Opticlean enzymes by Solvay Enzymes), Plasminogen Activator thrombosis, chronic inflammation, cancer marker Urokinase cancer marker General Protease screen, screening of protein formulations for the presence of proteases. Sensitive detection of DNA in solution and on blots utilizing thermophilic enzymes such as thermolysin which would enable the incorporation of the enzyme label during the DNA preparation procedure such as high temperature denaturation and PCR amplification. ______________________________________
See also generally Proteolytic Enzymes--A Practical Approach, IRL Press, pp. 233-40 (1989).